Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland.
Molecules. 2018 Feb 10;23(2):374. doi: 10.3390/molecules23020374.
Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet activation.
二磷酸腺苷（ADP）是主要的血小板激动剂，在血栓的形态变化、稳定性和生长中起重要作用。ADP 激活血小板与 G 蛋白偶联受体 P2Y1 和 P2Y12 有关。P2Y12 受体的药理阻断显著降低外周动脉疾病、心肌梗死、缺血性中风和血管死亡的风险。最近的研究表明，三种主要的黄酮木脂素化合物：水飞蓟宾、水飞蓟素和水飞蓟宁，可以抑制 ADP 诱导的血小板激活。因此，目前的工作旨在验证水飞蓟宾、水飞蓟素和水飞蓟宁对 ADP 诱导的生理血小板反应的影响，以及 P2Y12 依赖性细胞内信号转导的机制。我们评估了测试的黄酮木脂素对富含血小板的血浆（PRP）中 ADP 诱导的血小板聚集（使用透光比浊法）、纤维蛋白原粘附（使用静态方法）和 PF-4 分泌（使用 ELISA 法）的影响。此外，我们使用双标记流式细胞术方法估计血小板血管扩张刺激磷蛋白（VASP）磷酸化。我们证明了在用黄酮木脂素处理的样本中，血小板对 ADP 诱导的聚集、粘附纤维蛋白原和分泌 PF-4 的能力呈剂量依赖性降低。此外，我们观察到所有测试的黄酮木脂素都能够增加血小板样本中 VASP 的磷酸化，这与 P2Y12 受体抑制有关。所有这些分析表明，水飞蓟素和水飞蓟宾对 ADP 诱导的血小板激活具有最强的抑制作用，而水飞蓟宁也抑制 ADP 途径，但抑制作用较弱。本研究结果清楚地表明，水飞蓟宾、水飞蓟素和水飞蓟宁对 P2Y12 受体具有抑制作用，并阻断 ADP 诱导的血小板激活。
